Early phase evaluation of anticancer drugs has traditionally used toxicity (usually hematological) rather than efficacy end points to establish appropriate dosing schedules. To establish a biochemical efficacy end point for overcoming alkylguanine DNA alkyltransferase (AGT)-mediated tumor cell resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea, we performed a novel dose escalation clinical trial for the AGT-depleting agent 06-benzylguanine (BG). The dose of BG required to deplete AGT to undetectable levels (BMD(T) in sequential computed tomography-guided tumor tissue biopsies before BG and 18 h after BG was determined. Thirty patients received doses of BG ranging from 10 to 120 mg/m2 on the GCRC.